Thermo MK3 microplate reader
This experimental protocol uses Thermo Scientific's Multiskan MK3 microplate reader and Wellwash 4MK2 type plate washer (provided by Beijing Pingliyang Co., Ltd.), combined with Abraxis melamine detection kit, to test the antigen by competition method for liquid milk or milk powder. The melamine was tested and analyzed.
First, sample preparation
Pasteurized whole milk (liquid):
1) Dilute the milk sample with a dilution of 1:10, such as adding 100 μl of milk to a 900 μl dilution.
Infant formula:
1) Dissolve the milk powder in the diluent according to the instructions of the formula, such as dissolve 8.6 g of milk powder with 60 ml of the diluent.
2) Dilute the milk powder solution with a diluent at a ratio of 1:5
Second, sample testing
1) Reagent should be returned to room temperature (25 ± 2 ° C) before use.
2) Pipette 100 μl of standard (0, 20, 50, 100, 200 and 500 ppb) and the prepared sample into the corresponding microwell. Then, 50 μl of a melamine HRP enzyme label was added to each well.
3) Using the oscillation function of the MK3 microplate reader, shake the plate for 60 seconds and incubate for 30 minutes at room temperature.
4) After the incubation, transfer the microplate to the Wellwash 4MK2 plate washer for washing, add 250 μl of wash solution to each well, and wash the plate four times.
5) Add 100 μl of chromogenic substrate solution to each well and incubate for 20 minutes in the dark.
6) Add 100 μl of stop solution to each well to stop the color reaction.
7) The plate was read on a MK3 plate reader and the measurement wavelength was 450 nm.
Third, the results analysis
1) Based on the absorbance value of the 0 ppb standard (B0), the normalized standard absorbance B/B0 (B refers to the absorbance of the standard other than B0).
2) Draw a scatter plot with the B/B0 of the standard as the Y-axis coordinate and the corresponding standard concentration as the X-axis coordinate.
3) Straight line fitting the scatter plot. The concentration of the sample to be measured is calculated by the linear equation of the fitted curve.
4) If the absorbance of the sample is greater than the absorbance of the minimum standard, the concentration of the sample is <20 ppb; if the absorbance of the sample is less than the absorbance of the maximum standard, the concentration of the sample is >500 ppb.
5) Calculate the concentration of melamine in the final liquid milk or milk powder in combination with the dilution factor. For example, the dilution ratio of liquid milk is 10 times.
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